Oxidized Cell-Free DNA Rapidly Skews the Transcriptional Profile of Brain Cells toward Boosting Neurogenesis and Neuroplasticity.

Cell-free DNA (cfDNA) is liberated and accumulated in neural tissue due to cell damage. The oxidative and nitrosative stress in the brain that accompanies various pathological conditions has been shown to increase the oxidation of cellular and cell-free DNA. Whether the high concentration of non-oxidized and oxidized cfDNA may affect the transcriptome response of brain cells has not been studied. In the current work, we studied whether cfDNA fragments affect several key pathways, including neurogenesis, at the level of gene expression in brain cells. In the study, primary rat cerebellum cell cultures were used to assess the effects of oxidized and non-oxidized cfDNA on the expression of 91 genes in brain cells. We found that only oxidized cfDNA, not non-oxidized cfDNA, significantly altered the transcription in brain cells in 3 h. The pattern of change included all 10 upregulated genes (S100A8, S100A9, S100b, TrkB, Ngf, Pink1, Aqp4, Nmdar, Kcnk2, Mapk1) belonging to genes associated with neurogenesis and neuroplasticity. The expression of inflammatory and apoptosis genes, which oppose neurogenesis, decreased. The results show that the oxidized form of cfDNA positively regulates early gene expression of neurogenesis and neuroplasticity. At the same time, the question of whether chronic elevation of cfDNA concentration alters brain cells remains unexplored.

Oxidized cell-free DNA as a stress-signaling factor activating the chronic inflammatory process in patients with autism spectrum disorders.

BACKGROUND: Autism spectrum disorders (ASD) are known to be associated with an inflammatory process related to immune system dysfunction. This study's aim was to investigate the role of cell-free DNA in chronic inflammatory process in ASD patients. METHODS: The study included 133 ASD patients and 27 healthy controls. Sixty-two ASD patients were demonstrated to have mild-to-moderate disease severity (group I) and 71 individuals to have severe ASD (group II). Plasma cell-free (cf) DNA characteristics, plasma cytokine concentrations, expression of the genes for NFкB1 transcription factor and pro-inflammatory cytokines TNFα, IL-1ß and IL-8 in peripheral blood lymphocytes (PBL) of ASD patients, and unaffected controls were investigated. Additionally, in vitro experiments with oxidized DNA supplementation to PBL cultures derived from ASD patients and healthy controls were performed. RESULTS: The data indicates that ASD patients have demonstrated increased cfDNA concentration in their circulation. cfDNA of patients with severe ASD has been characterized by a high abundance of oxidative modification. Furthermore, ASD patients of both groups have shown elevated plasma cytokine (IL-1ß, IL-8, IL-17A) levels and heightened expression of genes for NFкB1 nuclear factor and pro-inflammatory cytokines TNFα, IL-1ß, and IL-8 in PBL. In vitro experiments have shown that NF-κB/cytokine mRNA expression profiles of ASD patient PBL treated with oxidized DNA fragments were significantly different from those of healthy controls. CONCLUSIONS: It may be proposed that oxidized cfDNA plays a role of stress-signaling factor activating the chronic inflammatory process in patients with ASD.

Altered recognition of antigen is a mechanism of CD8+ T cell tolerance in cancer.

Antigen-specific CD8+ T-cell tolerance, induced by myeloid-derived suppressor cells (MDSCs), is one of the main mechanisms of tumor escape. Using in vivo models, we show here that MDSCs directly disrupt the binding of specific peptide-major histocompatibility complex (pMHC) dimers to CD8-expressing T cells through nitration of tyrosines in a T-cell receptor (TCR)-CD8 complex. This process makes CD8-expressing T cells unable to bind pMHC and to respond to the specific peptide, although they retain their ability to respond to nonspecific stimulation. Nitration of TCR-CD8 is induced by MDSCs through hyperproduction of reactive oxygen species and peroxynitrite during direct cell-cell contact. Molecular modeling suggests specific sites of nitration that might affect the conformational flexibility of TCR-CD8 and its interaction with pMHC. These data identify a previously unknown mechanism of T-cell tolerance in cancer that is also pertinent to many pathological conditions associated with accumulation of MDSCs.

HEMOPERFUSION USING THE LPS-SELECTIVE MESOPOROUS POLYMERIC ADSORBENT IN SEPTIC SHOCK: A MULTICENTER RANDOMIZED CLINICAL TRIAL.

ABSTRACT: Extracorporeal hemoperfusion (EHP) may improve the course and outcomes of patients with septic shock by targeting cytokines or bacterial endotoxins (lipopolysaccharide [LPS]). Here, we present the results of a multicenter randomized controlled trial ( clinicaltrials.gov/ct2/show/NCT04827407 ) to assess the efficiency and safety of Efferon LPS hemoperfusion cartridges engineered for multimodal targeting LPS, host-derived cytokine, and damage-associated molecule pattern molecules. Patients with intra-abdominal sepsis (IAS) and septic shock (Sepsis-3) were subjected to EHP procedures (n = 38). Control patients with IAS and septic shock (n = 20) were treated using conventional protocols without EHP. The primary end point was resolution of septic shock. Secondary end points included MAP, vasopressor drug dose, partial pressure of arterial oxygen/fraction of inspired oxygen ratio, Sequential Organ Failure Assessment score, length of stay in the intensive care unit, and satisfaction with device use by a 5-point Likert scale. Clinical laboratory tests for a blood cells count, lactate and creatinine concentration, nephelometry test for C-reactive protein, immunochemiluminescent test for procalcitonin, and immunoenzyme analysis for IL-6 concentration were used to monitor the EHP effect versus the control group. Data were analyzed followed the intention-to-treat approach. Wilcoxon STATA 16.0 (StataCorp, College Station, TX) and Excel 2019 with XLStat 2019 add-in (Addinsoft, Paris, France) were used for statistical analysis of the results. The Fine and Gray method of competing risks was used to analyze the primary end point and other data representing the time to event. EHP resulted in a significant and rapid increase in MAP and partial pressure arterial oxygen/fraction of inspired oxygen ratio, progressive decline in norepinephrine doses, and multiorgan deficiency, as evaluated by Sequential Organ Failure Assessment scores. Importantly, EHP led to significantly rapid cumulative mechanical ventilation weaning compared with the control group (subdistribution hazard ratio, 2.5; P = 0.037). Early 3-day mortality was significantly reduced in the Efferon LPS versus control group; however, no significant improvements in survival in 14 and 28 days were revealed. Laboratory tests showed rapidly decreased levels of LPS, procalcitonin, C-reactive protein, IL-6, creatinine, leukocytes, and neutrophils only in the Efferon LPS group. Results demonstrate that EHP with Efferon LPS is a safe procedure to abrogate septic shock and normalize clinical and pathogenically relevant biomarkers in patients with IAS.

Association of NEF2L2 Rs35652124 Polymorphism with Nrf2 Induction and Genotoxic Stress Biomarkers in Autism.

Increased oxidative/genotoxic stress is known to impact the pathophysiology of ASD (autism spectrum disorder). Clinical studies, however, reported limited, heterogeneous but promising responses to treatment with antioxidant remedies. We determined whether the functional polymorphism of the Nrf2 gene, master regulator of anti-oxidant adaptive reactions to genotoxic stress, links to the genotoxic stress responses and to an in vitro effect of a NRF2 inductor in ASD children. Oxidative stress biomarkers, adaptive responses to genotoxic/oxidative stress, levels of master antioxidant regulator Nrf2 and its active form pNrf2 before and after inducing by dimethyl fumarate (DMF), and promotor rs35652124 polymorphism of NFE2L2 gene encoding Nrf2 were studied in children with ASD (n = 179). Controls included healthy adults (n = 101). Adaptive responses to genotoxicity as indicated by H2AX and cytoprotection by NRF2 contents positively correlated in ASD children with a Spearman coefficient of R = 0.479 in T+, but not CC genotypes. ASD children with NRF2 rs35652124 CC genotype demonstrated significantly higher H2AX content (0.652 vs. 0.499 in T+) and pNrf2 induction by DMF, lowered 8-oxo-dG concentration in plasma and higher cfDNA/plasma nuclease activity ratio. Our pilot findings suggest that in ASD children the NEF2L2 rs35652124 polymorphism impacts adaptive responses that may potentially link to ASD severity. Our data warrant further studies to reveal the potential for NEF2L2 genotype-specific and age-dependent repurposing of DMF and/or other NRF2-inducing drugs.

FIB-DIC Residual Stress Evaluation in Shot Peened VT6 Alloy Validated by X-ray Diffraction and Laser Speckle Interferometry.

Ga-ion micro-ring-core FIB-DIC evaluation of residual stresses in shot peened VT6 (Ti-6Al-4V) alloy was carried out and cross-validated against other non-destructive and semi-destructive residual stresses evaluation techniques, namely, the conventional sin2ψ X-ray diffraction and mechanical hole drilling. The Korsunsky FIB-DIC method of Ga-ion beam micro-ring-core milling within FIB-SEM with Digital Image Correlation (DIC) deformation analysis delivered spatial resolution down to a few micrometers, while the mechanical drilling of circular holes of ~2 mm diameter with laser speckle interferometry monitoring of strains gave a rough spatial resolution of a few millimeters. Good agreement was also found with the X-ray diffraction estimates of residual stress variation profiles as a function of depth. These results demonstrate that FIB-DIC provides rich information down to the micron scale, it also allows reliable estimation of macro-scale residual stresses.

Peroxynitrite in the tumor microenvironment changes the profile of antigens allowing escape from cancer immunotherapy.

Cancer immunotherapy often depends on recognition of peptide epitopes by cytotoxic T lymphocytes (CTLs). The tumor microenvironment (TME) is enriched for peroxynitrite (PNT), a potent oxidant produced by infiltrating myeloid cells and some tumor cells. We demonstrate that PNT alters the profile of MHC class I bound peptides presented on tumor cells. Only CTLs specific for PNT-resistant peptides have a strong antitumor effect in vivo, whereas CTLs specific for PNT-sensitive peptides are not effective. Therapeutic targeting of PNT in mice reduces resistance of tumor cells to CTLs. Melanoma patients with low PNT activity in their tumors demonstrate a better clinical response to immunotherapy than patients with high PNT activity. Our data suggest that intratumoral PNT activity should be considered for the design of neoantigen-based therapy and also may be an important immunotherapeutic target.

Effect of Xenon Treatment on Gene Expression in Brain Tissue after Traumatic Brain Injury in Rats.

The overactivation of inflammatory pathways and/or a deficiency of neuroplasticity may result in the delayed recovery of neural function in traumatic brain injury (TBI). A promising approach to protecting the brain tissue in TBI is xenon (Xe) treatment. However, xenon's mechanisms of action remain poorly clarified. In this study, the early-onset expression of 91 target genes was investigated in the damaged and in the contralateral brain areas (sensorimotor cortex region) 6 and 24 h after injury in a TBI rat model. The expression of genes involved in inflammation, oxidation, antioxidation, neurogenesis and neuroplasticity, apoptosis, DNA repair, autophagy, and mitophagy was assessed. The animals inhaled a gas mixture containing xenon and oxygen (ϕXe = 70%; ϕO2 25-30% 60 min) 15-30 min after TBI. The data showed that, in the contralateral area, xenon treatment induced the expression of stress genes (Irf1, Hmox1, S100A8, and S100A9). In the damaged area, a trend towards lower expression of the inflammatory gene Irf1 was observed. Thus, our results suggest that xenon exerts a mild stressor effect in healthy brain tissue and has a tendency to decrease the inflammation following damage, which might contribute to reducing the damage and activating the early compensatory processes in the brain post-TBI.

Udp-glucose dehydrogenase as a novel field-specific candidate biomarker of prostate cancer.

Uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) catalyzes the oxidation of UDP-glucose to yield UDP-glucuronic acid, a precursor for synthesis of glycosaminoglycans and proteoglycans that promote aggressive prostate cancer (PC) progression. The purpose of our study was to determine if the UGDH expression in normal appearing acini (NAA) from cancerous glands is a candidate biomarker for PC field disease/effect assayed by quantitative fluorescence imaging analysis (QFIA). A polyclonal antibody to UGDH was titrated to saturation binding and fluorescent microscopic images acquired from fixed, paraffin-embedded tissue slices were quantitatively analyzed. Specificity of the assay was confirmed by Western blot analysis and competitive inhibition of tissue labeling with the recombinant UGDH. Reproducibility of the UGDH measurements was high within and across analytical runs. Quantification of UGDH by QFIA and Reverse-Phase Protein Array analysis were strongly correlated (r = 0.97), validating the QFIA measurements. Analysis of cancerous acini (CA) and NAA from PC patients vs. normal acini (NA) from noncancerous controls (32 matched pairs) revealed significant (p < 0.01) differences, with CA (increased) vs. NA, NAA (decreased) vs. NA and CA (increased) vs. NAA. Areas under the Receiver Operating Characteristic curves were 0.68 (95% CI: 0.59-0.83) for NAA and 0.71 (95% CI: 0.59-0.83) for CA (both vs. NA). These results support the UGDH content in prostatic acini as a novel candidate biomarker that may complement the development of a multi-biomarker panel for detecting PC within the tumor adjacent field on a histologically normal biopsy specimen.

Changes of KEAP1/NRF2 and IKB/NF-κB Expression Levels Induced by Cell-Free DNA in Different Cell Types.

Cell-free DNA (cfDNA) is a circulating DNA of nuclear and mitochondrial origin mainly derived from dying cells. Recent studies have shown that cfDNA is a stress signaling DAMP (damage-associated molecular pattern) molecule. We report here that the expression profiles of cfDNA-induced factors NRF2 and NF-κB are distinct depending on the target cell's type and the GC-content and oxidation rate of the cfDNA. Stem cells (MSC) have shown higher expression of NRF2 without inflammation in response to cfDNA. In contrast, inflammatory response launched by NF-κB was dominant in differentiated cells HUVEC, MCF7, and fibroblasts, with a possibility of transition to massive apoptosis. In each cell type examined, the response for oxidized cfDNA was more acute with higher peak intensity and faster resolution than that for nonoxidized cfDNA. GC-rich nonoxidized cfDNA evoked a weaker and prolonged response with proinflammatory component (NF-κB) as predominant. The exploration of apoptosis rates after adding cfDNA showed that cfDNA with moderately increased GC-content and lightly oxidized DNA promoted cell survival in a hormetic manner. Novel potential therapeutic approaches are proposed, which depend on the current cfDNA content: either preconditioning with low doses of cfDNA before a planned adverse impact or eliminating (binding, etc.) cfDNA when its content has already become high.

Hematopoietic progenitor cell mobilization in mice by sustained delivery of granulocyte colony-stimulating factor.

The objective of these studies was to determine the effect of sustained delivery of growth factors (GFs) on hematopoietic progenitor cells (HPCs) in mice. In these studies, granulocyte colony-stimulating factor (G-CSF) was administered using the poloxamer-based matrix, ProGelz (PG) and G-CSF, and pharmacokinetics (PKs) and HPC mobilization was assessed. A single injection of G-CSF formulated in PG (17% poloxamer-407 and 5% hydroxypropyl methylcellulose [HPMC]) administered to BALB/c mice mobilized HPC significantly more rapidly to the spleen, but not the blood, than multiple injections of saline-formulated G-CSF. Two days after a single injection of PG G-CSF, the frequency of colony-forming unit-culture (CFU-c) in the spleen was increased 289-fold compared with an 8-fold increase after 2 days of twice-daily injections of saline-formulated G-CSF. Indeed, 4 days of twice-daily G-CSF injections were required to achieve the same level of HPC mobilization. In contrast, a similar mobilization of HPC to the blood was observed between PG and saline-formulated G-CSF. The mechanism for the accelerated and increased mobilization to the spleen by the PG-formulation of G-CSF is due, in part, to its increased bioavailability (>1.5-fold), T(max) (6-fold), and prolonged elimination (Tbeta) half-life (>3-fold) as compared with a saline formulation. In addition, we observed a more rapid trafficking of the PG G-CSF to the marrow, which could also facilitate mobilization.

Murine mammary adenocarcinoma cells transfected with p53 and/or Flt3L induce antitumor immune responses.

Transfection of tumors with tumor-associated antigens (Ags) or cytokines can increase immunogenicity and slow down tumor growth. However, the effect of cotransfection with genes that encode a tumor-associated Ag, such as the tumor suppressor gene p53, and a cytokine has been rarely investigated. We report that transfection of 4T1 mammary tumor cells (p53-null) with the dendritic cell (DC) growth factor, fms-like tyrosine kinase 3 ligand (Flt3L), significantly delayed their growth in vivo, resulting in the rejection of 100% of the tumors formed by injection of tumor cells cotransfected with Flt3L and p53. Immunization with irradiated 4T1 cells transfected with Flt3L induced DC infiltration of the immunization site and significantly increased the antitumor T-cell responses. Further, immunization with irradiated 4T1 cells cotransfected with p53 and Flt3L significantly increased p53-specific immune responses, as compared to vaccination with 4T1 cells transfected with either Flt3L or p53 alone. These responses included increased activity against clone 66 (Cl-66), a sister tumor to 4T1 with high murine mutant p53 expression levels. Challenge with Cl-66 revealed that immunization with irradiated 4T1-Flt3L-p53 cells significantly slowed growth, prolonged survival, and resulted in complete remissions. Further, immunization with irradiated 4T1-Flt3L also slowed Cl-66 growth, although to a lesser extent than 4T1-Flt3L-p53. We suggest that immunization with DCs transfected with the Flt3L transgene and a tumor Ag may potentially heighten T-cell responses and therapeutic activity.

T cells recognize PD(N/T)R motif common in a variable number of tandem repeat and degenerate repeat sequences of MUC1.

The tumor-associated antigen MUC1 is a transmembrane glycoprotein, which is overexpressed in human carcinomas. Peptide epitopes, containing the PDTR fragment from the variable number of tandem repeat (VNTR) domains of MUC1 have been found to be immunodominant in T-cell and B-cell responses. However, little is known about the immunogenicity and specificity of T-cell epitopes from other regions of MUC1 that may also participate in immune responses against tumors. In this study, the combination of immunoinformatics, molecular modeling and a vaccine adjuvant strategy were used to predict and describe a novel T-cell epitope, SAPDNRPAL, located within the degenerate tandem repeat of MUC1. This peptide possesses structural similarity to both VNTR-derived SAPDTRPAP and Sendai virus peptide FAPGNYPAL, which are known to induce cytotoxic T lymphocytes (CTL). We found that SAPDNRPAL had a higher affinity for mouse H-D(b), H-2K(b) and human HLA-A2 molecules than SAPDTRPAP. A chimeric peptide (CP) containing SAPDNRPAL and an adjuvant C5a-derived decapeptide induced epitope-specific type 1 T cells in human MUC1 transgenic mice (ELISPOT). Mice that received dendritic cells (DC) pulsed with the CP or a 25-mer peptide containing the SAPDNRPAL sequence showed increased frequencies of SAPDNRPAL- and SAPDTRPAP-specific interferon-gamma producing T cells. PDTR-specific antibody 214D4 reacted with both SAPDNRPAL and SAPDTRPAP (ELISA). Altogether, our data suggest that the degenerate MUC1 repeat sequence contains the immunogenic T-cell epitope SAPDNRPAL, which is cross-reactive with the VNTR-derived peptide SAPDTRPAP. We suggest that the use of immunogenic PDNR-containing epitope(s) in vaccine strategies could be beneficial for developing increased, PD(N/T)R motif-specific T-cell responses against tumors expressing MUC1.

Full-length dominant-negative survivin for cancer immunotherapy.

PURPOSE: The goal of this study is to investigate the possible utility of dendritic cells (DCs) transduced with the human full-length dominant-negative survivin for cancer immunotherapy. EXPERIMENTAL DESIGN: Mononuclear cells were collected from HLA-A2-positive healthy volunteers and patients with prostate cancer. DCs were transduced with an adenoviral vector containing a full-length, dominant-negative survivin gene. After three rounds of stimulation, the T-cell response against three different survivin-derived HLA-A2-matching peptides was tested in IFN-gamma enzyme-linked immunospot and CTL assays. RESULTS: Seven of eight healthy volunteers and cancer patients showed a significant response to at least two different survivin-derived epitopes in the enzyme-linked immunospot assay. One patient responded to only one peptide. All four healthy volunteers and two of three patients tested demonstrated a specific CTL response against T2 target cells loaded with one survivin-derived epitope. Two donors and two patients had a significant CTL response against two different epitopes. Significant cytotoxic activity was seen against HLA-A2-positive MCF-7 tumor cells that express survivin. That response was specific for survivin and was MHC class I restricted. Because survivin is expressed in CD34(+) hematopoietic progenitor cells (HPCs), we tested whether the antisurvivin CTLs can recognize normal HPCs. The incubation of survivin-specific CTLs with CD34(+) cells did not significantly decrease the colony-forming ability of HPCs. CONCLUSIONS: DCs transduced with dominant-negative survivin induce potent survivin-specific CTL responses able to recognize and kill tumor cells. This response does not significantly affect HPCs. Thus, this study may provide rationale for immunotherapeutic clinical trials using a DC vaccine transduced with the dominant-negative survivin.

Modeling and simulation of a low-grade urinary bladder carcinoma.

In this work, we present a mathematical model of the initiation and progression of a low-grade urinary bladder carcinoma. We simulate the crucial processes affecting tumor growth, such as oxygen diffusion, carcinogen penetration, and angiogenesis, within the framework of the urothelial cell dynamics. The cell dynamics are modeled using the discrete technique of cellular automata, while the continuous processes of carcinogen penetration and oxygen diffusion are described by nonlinear diffusion-absorption equations. As the availability of oxygen is necessary for tumor progression, processes of oxygen transport to the tumor growth site seem most important. Our model yields a theoretical insight into the main stages of development and growth of urinary bladder carcinoma with emphasis on the two most common types: bladder polyps and carcinoma in situ. Analysis of histological structure of bladder tumor is important to avoid misdiagnosis and wrong treatment. We expect our model to be a valuable tool in the study of bladder cancer progression due to the exposure to carcinogens and the oxygen dependent expression of genes promoting tumor growth. Our numerical simulations have good qualitative agreement with in vivo results reported in the corresponding medical literature.

Tumor escape from immune response: mechanisms and targets of activity.

Immune system plays an important role in control of tumor progression. Effective antitumor immune response depends on close interaction of several elements of immune system. They include antigen-presenting cells, different subsets of T cells, B cells and NK cells. However, tumor cells developed a number of mechanisms to escape recognition and elimination by immune system. In this review we will discuss these mechanisms and address possible approaches to correct them.

Flt3 ligand augmentation of T cell mitogenesis and expansion of type 1 effector/memory T cells.

Herein we report mechanisms whereby Flt3 ligand (FL) augments steady state T cell activity in addition to the expansion of dendritic cells (DCs). We demonstrate that in vivo administration of FL increases the frequency and absolute number of effector/memory T cells and preferentially expands T cells that express a type-1 cytokine phenotype. In addition, FL enhances T cell proliferative responses to Concanavalin A that directly correlated with increased frequencies in effector/memory T cells and expansion of lymphoid-derived (type 1) DCs (DC1s). Together, these data demonstrate that mechanisms of FL-induced T cell regulation include not only the expansion of DC subsets, but also the preferential expansion of type 1 -effector/memory T cell populations, and suggest multiple mechanisms of action for FL as a vaccine adjuvant and as a therapeutic modality.

Cellular immunotherapy study of prostate cancer patients and resulting IgG responses to peptide epitopes predicted from prostate tumor-associated autoantigens.

The immunogenicity of a cellular immunotherapy using genetically modified vaccines to express α(1,3)galactosyl epitopes (αGal) was evaluated in advanced prostate cancer (PC) patients. In this dose escalation phase I study, we report safety, feasibility, and immunologic data of an immunotherapy composed of 2 human PC cell lines engineered to express αGal epitopes (HyperAcute-Prostate, HAP, NewLink Genetics). Eight patients received up to 12 biweekly vaccinations with HAP. Enrolled patients (aged range, 53-85 y) had American Joint Committee on Cancer stage IV, any T, any N, M1, Eastern Cooperative Oncology Group PS≤2, at least 1 prior hormonal treatment and <3 prior chemotherapies, adequate bone marrow and organ function, and albumin ≥3.0 g/dL. Serum IgG antibodies to synthetic peptides overexpressed in PC were determined by enzyme-linked immunosorbent assay. Results indicate that HAP immunotherapy induced humoral immune responses to autoantigens in 2 of 8 patients. These patients developed IgG antibody to multiple epitopes overexpressed in PC after immunization. These responding patients received higher doses of the immunotherapy suggesting a dose response. Two immunogenic proteins (prostate-specific membrane antigen, hepsin) belong to the extracellular molecules family participating in malignant cell invasion. Median overall survival for patients was 25.1 months with 1 patient surviving over 70 months with stable PSA and bone metastasis before expiring of other causes. Three of 8 patients showed PSA stabilization (>100 d). In conclusion, HAP immunotherapy induces IgG responses to epitopes from autoantigens overexpressed in PC suggesting dose-dependent effect. HAP represents a viable immunotherapy approach to induce immune responses against tumor cells and may provide clinical benefit with minimal toxicity.

Tumor-infiltrating myeloid cells induce tumor cell resistance to cytotoxic T cells in mice.

Cancer immunotherapeutic approaches induce tumor-specific immune responses, in particular CTL responses, in many patients treated. However, such approaches are clinically beneficial to only a few patients. We set out to investigate one possible explanation for the failure of CTLs to eliminate tumors, specifically, the concept that this failure is not dependent on inhibition of T cell function. In a previous study, we found that in mice, myeloid-derived suppressor cells (MDSCs) are a source of the free radical peroxynitrite (PNT). Here, we show that pre-treatment of mouse and human tumor cells with PNT or with MDSCs inhibits binding of processed peptides to tumor cell-associated MHC, and as a result, tumor cells become resistant to antigen-specific CTLs. This effect was abrogated in MDSCs treated with a PNT inhibitor. In a mouse model of tumor-associated inflammation in which the antitumor effects of antigen-specific CTLs are eradicated by expression of IL-1ß in the tumor cells, we determined that therapeutic failure was not caused by more profound suppression of CTLs by IL-1ß-expressing tumors than tumors not expressing this proinflammatory cytokine. Rather, therapeutic failure was a result of the presence of PNT. Clinical relevance for these data was suggested by the observation that myeloid cells were the predominant source of PNT in human lung, pancreatic, and breast cancer samples. Our data therefore suggest what we believe to be a novel mechanism of MDSC-mediated tumor cell resistance to CTLs.